EP3712263A1 - Synthase d'acide gras, ses inhibiteurs et modifications et son utilisation - Google Patents

Synthase d'acide gras, ses inhibiteurs et modifications et son utilisation Download PDF

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EP3712263A1
EP3712263A1 EP19163958.2A EP19163958A EP3712263A1 EP 3712263 A1 EP3712263 A1 EP 3712263A1 EP 19163958 A EP19163958 A EP 19163958A EP 3712263 A1 EP3712263 A1 EP 3712263A1
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EP
European Patent Office
Prior art keywords
protein complex
fas
fas protein
mpt
enoylreductase
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EP19163958.2A
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German (de)
English (en)
Inventor
Ashwin CHARI
Holger Stark
Kashish SINGH
Benjamin Moritz GRAF
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Priority to EP19163958.2A priority Critical patent/EP3712263A1/fr
Priority to CN202080037069.5A priority patent/CN113853431A/zh
Priority to JP2021555473A priority patent/JP2022525203A/ja
Priority to CA3132089A priority patent/CA3132089A1/fr
Priority to BR112021017141A priority patent/BR112021017141A2/pt
Priority to EP20711942.1A priority patent/EP3942027A2/fr
Priority to KR1020217032202A priority patent/KR20210141537A/ko
Priority to PCT/EP2020/057741 priority patent/WO2020188074A2/fr
Priority to SG11202109475T priority patent/SG11202109475TA/en
Priority to US17/440,425 priority patent/US20220154181A1/en
Publication of EP3712263A1 publication Critical patent/EP3712263A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
    • G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
    • G01N23/225—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion
    • G01N23/2251—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion using incident electron beams, e.g. scanning electron microscopy [SEM]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01086—Fatty-acyl-CoA synthase (2.3.1.86)

Definitions

  • the present invention relates to new recombinant polypeptides derived from the newly identified gamma subunit of the fatty acid synthase protein complex.
  • a fatty acid synthase protein complex comprising these new recombinant polypeptides are disclosed as well as nucleic acid molecules encoding these polypeptides.
  • host cells containing the nucleic acid molecule encoding the polypeptides according to the present invention or expressing the polypeptide according to the present invention are described.
  • an isolated fatty acid synthase protein complex is disclosed containing the newly identified gamma subunit thereof.
  • the fatty acid synthase is a multi-enzyme protein that catalyzes fatty acid synthesis. So far it is described that said enzyme in yeast is an enzymatic system composed of two multifunctional subunits in which substrates are handed from one functional domain to the next, by means of an ACP domain encoded by the alpha subunit.
  • the fatty acid synthase (FAS) catalyzes the synthesis of palmitate from acetyl-CoA and malonyl-CoA in the presence of NADPH.
  • FAS type I system utilise a single or two large, multifunctional polypeptide and are common to both animals and fungi.
  • a type I fatty acid synthase system can also be found in a subgroup of bacteria, namely, the corynebacteria, mycobacteria, and nocardia. In these bacteria, the FAS I system produces palmitic acid and cooperates with the FAS II system described below to produce a greater diversity of lipid products.
  • the FAS type II is found in archaea and bacteria, and is characterized by the use of discrete, monofunctional enzymes for fatty acid synthesis. Inhibitors of this pathway of FAS II are being investigated as possible antibiotics.
  • the yeast fatty acid synthase as a type I FAS is described as a 2.6 MDa barrel-shaped complex and should be composed of two unique multifunctional subunits, ⁇ and ⁇ . It is described that said ⁇ and ⁇ units are arranged in an ⁇ 6 ⁇ 6 structure.
  • the catalytic activities of this enzyme complex involve a coordination system of enzymatic reactions between the ⁇ - and ⁇ -subunits. It is submitted that the enzyme complex consists of six functional centers for fatty acid synthesis.
  • the FAS is regarded as a suitable target for treating various diseases including cancer since the FAS is upregulated e. g.in breast cancer etc.
  • the ⁇ -subunit contains the ketoreductase (KR), ketosynthase (KS) and phosphopantetheinyl transferase (PPT) domains, the ⁇ -subunit harbors the acetyltransferase (AT), enoylreductase (ER) and dehydratase (DH) domains.
  • KR ketoreductase
  • KS ketosynthase
  • PPT phosphopantetheinyl transferase
  • AT acetyltransferase
  • ER enoylreductase
  • DH dehydratase
  • the malonyl/palmitoyl transferase (MPT) domain is mostly formed by the very C-terminal part of the ⁇ -subunit, but is completed by the N-terminus of the ⁇ -subunit.
  • ACP acyl carrier protein
  • fatty acids have emerged as valuable platform compounds for the biotechnological production of fine chemicals and biofuels from renewable resources. Indeed, type I FAS megaenzymes, including those with the architecture of yeast FAS, have been successfully subjected to protein engineering to generate e.g.
  • the present invention relates to a recombinant polypeptide composed of amino acids 1 to 132 of SEQ ID No. 1 or a functional fragment thereof, or a homolog thereof. Further, a recombinant polypeptide is provided comprising the amino acids of SEQ ID No. 1 containing further an insert of at least 20 amino acids, like 25 amino acids or a functional fragment thereof, or a homolog thereof.
  • polypeptide consisting of
  • fatty acid synthase protein complex comprising the recombinant polypeptide as described herein is disclosed.
  • nucleic acid molecules encoding the polypeptides according to the present invention as well as host cells containing said nucleic acid molecules and/or expressing the polypeptides according to the present invention are described.
  • an isolated fatty acid synthase protein complex containing the described ⁇ - and ⁇ -subunits as well as the polypeptide of SEQ ID No. 1 is claimed.
  • Said isolated fatty acid synthase protein complex is obtainable by a method comprising the steps of
  • KR ketoredutase
  • ER enoylreductase
  • MTT malonyl/palmitoyl transferase
  • recombinant polypeptides are provided.
  • a recombinant polypeptide is provided wherein said polypeptide is composed of amino acids 1 to 132 of SEQ ID No. 1 or a functional fragment thereof, or a homologue thereof.
  • the term "functional fragment thereof” refers to polypeptides having the same functionality as the polypeptide or the nucleic acid molecule, respectively. That is, the functional fragment may contain a deletion or substitution of at least one amino acid residue or at least one nucleic acid residue.
  • polypeptides according to the present invention include fragments also known as derivatives wherein single amino acid molecules may be substituted with a different amino acid residue.
  • the amino acid at position 2 may be a serine instead of a cysteine.
  • the single substitutions include conservative amino acid substitutions. The skilled person is well aware of suitable substitutions without altering significantly the activity of the recombinant polypeptide.
  • homolog thereof refers to recombinant polypeptides as claimed herein being derived from other species, in particular, belonging to other yeast species as the y-unit described herein of SEQ ID No. 1 obtained from Saccharomyces cerevisiae. Homologs thereof include the same molecule from other saccharomyces species as well as from other eukaryotic species in particular yeast and bacterial species like candida, aspergillus, yarrowia, penicillum, Rhodospiridium, mycobacteria, chaetomium.
  • recombinant refers to polypeptides not being present in nature or which have been not isolated from nature before.
  • a candidate compound refers to a molecule being a small molecule, a chemical or biological molecule, in particular, being composed or comprising amino acid residues.
  • crystal refers to a supra-molecular assembly of any given single molecule or molecule species in a spatially repetitive manner.
  • the molecule or molecular species can be defined as any chemical moiety whether atoms are connected by covalent bonds and/or non-covalent bonds.
  • osmolyte refers to compounds affecting osmosis. Examples include but are not restricted to glycerol, sucrose, sugars in general, trimethylamine-N-oxide (TMAO) and ethylene glycol.
  • TMAO trimethylamine-N-oxide
  • polymer with zero net charge refers to a polymer, which is built up by the consecutive covalent attachment of a repeating chemical unit in the size range of hundreds of Daltons.
  • the definition of zero net charge is satisfied by the properties of the repeating unit, which ideally contains no electronic charge (neither positive nor negative). If electronic charges are present, they should appear in a balanced manner, i.e. positive charges with a value of two should be counter-balanced by negative charges with a value of two.
  • Such molecules with counter-balanced electronic charges are referred to as zwitterionic species or chemicals.
  • non-ionic polymer refers to a molecule, which is built up by the consecutive covalent attachment of a repeating chemical unit in the size range of hundreds of Daltons. When the chemical unit contains no electronic charge, the resulting polymer is referred to as a non-ionic polymer.
  • non-covalent bonds include but are not restricted to hydrogen-bonds, electrostatic interactions and van der Waals forces.
  • the relation of one molecule in a crystal to another can be described by strict symmetry rules which are based on the Bravais lattices.
  • the term "inhibitor” refers to a compound, including a biological or chemical entity capable of reducing a biological activity.
  • the inhibitor is a biological or chemical molecule which reduces the ketoreductase activity, enoylreductase activity and/or MPT activity.
  • an inhibitor is an entity which reduces the "normal" activity of said biological entity at least ten fold, like at least twenty fold, in particular, at least fifty fold.
  • FAS protein complex refers to the FAS I protein complex.
  • FAS protein complex refers to a protein complex being composed of at least three moieties, namely, the ⁇ -subunit, the ⁇ -subunit of the FAS protein complex of FAS I and a third unit, e.g. the candidate compound, a polypeptide as defined herein, including the gamma-subunit of SEQ ID No. 1 or fragments thereof, or homologs thereof.
  • polypeptide of SEQ ID No. 1 represents the TMA 17 molecule from Saccharomyces cerevisiae. So far, said polypeptide has been described as being induced upon stress and should be associated with proteasome assembly.
  • the present inventors recognized that the recombinant polypeptide composed of amino acids 1 to 132 of SEQ ID No. 1 or a functional fragment thereof, or a homolog thereof when being present in the FAS complex demonstrates higher ketoreductase activity compared to the FAS complex containing the full amino acid sequence of SEQ ID No. 1. That is, the present inventors recognized that the amino acids 133 to 150 of SEQ ID No. 1 represents the interacting sequence with the ketoreductase in the FAS protein complex. As demonstrated below, the presence of the full length polypeptide of SEQ ID No. 1 has regulatory roles in the activity of various enzymes present in the FAS protein complex.
  • a recombinant polypeptide comprising the amino acids of SEQ ID No. 1 further containing an insert of at least 15, like at least 20, like at least 23, 25, 28, 30 or more amino acids or a functional fragment thereof or a homolog thereof.
  • the polypeptide is a polypeptide containing said insert being composed from the N-terminus to the C-terminus of
  • the insert after any one of the amino acid 112 of SEQ ID No. 1 and before SEQ ID No. 132 of SEQ ID No. 1 may be particularly beneficial to avoid any negative influence on interacting properties of the remaining amino acids.
  • the recombinant polypeptide described include a polypeptide wherein in the second unit composed of amino acids 113 to 132 containing said insert, at least one or all of the amino acids 113 to 132 may be absent.
  • the insert present in the recombinant polypeptide according to the present invention is an enzyme.
  • the enzyme is selected from a desaturase, thioesterase, oxygenase, hydroxylase, cyclogenase, fatty acid lyase.
  • the insert present in the polypeptide according to the present invention may have a size of at most 60 kDa, like at most 50 kDa, for example 40 kDa, 35 kDa, 30 kDa, at most 25 kDa, at most 20 kDa.
  • the insert has at least a size as mentioned above, namely of at least 15 amino acids.
  • the size of the insert is at least 2 kDa, like at least 2,5 kDa, like at least 3 kDa.
  • polypeptide consist of
  • the amino acids of SEQ ID No. 2 represent the interacting interface with the ketoreductase present in the FAS protein complex.
  • the amino acids of SEQ ID No. 2 represents the amino acids present in the gamma-unit interacting with the enoylreductase and the amino acids of SEQ ID No. 4 represents the amino acid of the gamma-unit interacting with the MPT of the FAS protein complex.
  • polypeptide as defined above referring to SEQ ID Nos. 2, 3 and 4 represents interacting and, thus, regulating moieties with the respective enzyme activity present in the other subunits of the FAS protein complex.
  • the gamma-subunit also known as TMA 17, has various interacting sites with the other subunits of the FAS protein complex, namely of the ⁇ - and ⁇ -subunit.
  • the present inventions provide a fatty acid synthase protein complex comprising the recombinant polypeptide according to the present invention.
  • the fatty acid synthase protein complex according to the present invention contains ⁇ - and ⁇ -subunits as described in the art.
  • the present inventors identify surprisingly a further subunit present in the FAS protein complex (FAS I), namely, the Tma17 polypeptide previously known to interact with the proteasome or ribosome only.
  • the fatty acid synthase protein complex containing the recombinant polypeptide as described herein for use in the production of fatty acid and biofuels in particular, for use in the biological production of free fatty acids, keto-fatty acids, fatty acids alcohol, cyclic fatty acids and elongated fatty acids. That is, in particular using the recombinant polypeptide as defined in anyone of claims 2 to 5 containing the insert it is possible to introduce further enzymatic activities into the described sequence. Said enzymatic activities allow to act on the fatty acids, thus, changing further the product of the FAS protein complex.
  • a polypeptide according to the present invention having in inhibitory activity on at least one of the enzymes present on the FAS protein complex, one of the steps of the fatty acid synthesis conducted therein may be inhibited, thus, changing the product obtained.
  • the present invention relates to a nucleic acid molecule encoding a polypeptide according to the present invention.
  • the nucleic acid molecule may be composed of nucleic acids known in the art including DNA, RNA, or other molecules like PNA etc.
  • vectors are provided comprising the nucleic acid sequence according to the present invention encoding the peptide as described. The skilled person is well aware of suitable vector system and vectors, in particular, vectors allowing transfection und transduction of eukaryotic cells including transformation and recombination into the yeast genome.
  • the present inventions relates to a host cell containing the nucleic acid molecule according to the present invention and/or expressing a polypeptide according to the present invention.
  • the host cell is a microorganism containing a type I fatty acid synthase (FAS I), like yeast.
  • the present invention relates to an isolated fatty acid synthase protein complex obtainable by a method comprising the steps of:
  • the method allows to isolate the FAS protein complex including the newly identified gamma-subunit as described herein.
  • the present inventors conducting the method described in WO 2011/211775 A1 obtained a FAS protein complex containing a third subunit.
  • the subunit is the Tma17 polypeptide.
  • reduction of cysteines ist performed by the use of phoshine based reducing agents, like tricarboxyerthylphosphine (TCEP).
  • TCEP tricarboxyerthylphosphine
  • the density gradient is performed at room temperature, like at 18°C to 22°C.
  • the salt concentration (NaCl and KCl in combination) during the purification steps is maintained below 150 mM to minimize dissociation of the gamma-unit.
  • the present invention relates to a method for determining the suitability of a candidate compound capable of inhibiting a ketoreductase, in particular, the ketoreductase present in the FAS protein complex, comprising the steps of:
  • the present invention relates to a method for determining the suitability of a candidate compound capable of inhibiting an enoylreductase, in particular, the enoylreductase present in the FAS protein complex, comprising the steps of:
  • the present invention provides a method for the suitability of a candidate compound capable of inhibiting an malonyl/palmitoyl transferase (MPT), in particular, the MPT present in the FAS protein complex, comprising the steps of:
  • the methods described for identifying candidate compound capable of inhibiting the respective enzyme include either a crystallization of said enzyme or protein complex containing said enzyme or alternatively or in combination examining said candidate compound interacting with the respective enzyme by cryo-electron microscopy.
  • the candidate compound may be a compound which inhibit at least two of the enzymes mentioned, for example, inhibiting both the ketoreductase and the enoylreductase.
  • candidate compounds may be obtained from peptide libraries containing the mentioned inhibitory peptide sequences as described herein whereby these sequences are randomized by random mutation or site-directed mutation.
  • Candidate compounds binding to the enzyme in question in particular, one of the enzymes ketoreductase, enoylreductase or MPT are selected and introduced into the gamma-subunit, thus, allowing to determine the inhibitory activity on the respective enzyme.
  • the candidate compounds also include compounds stabilizing the inhibitory interference of the natural inhibitory unit of the gamma-subunit.
  • the skilled person is well aware of libraries and methods to determine usefulness of the candidate compunds.
  • a method for the design of an inhibitor of the enzyme activity present in the FAS protein complex is identified. Namely, a method for the design of an inhibitor of the ketoreductase activity, in particular, ketoreductase activity in the FAS protein complex comprising step of
  • a method for the design of an inhibitor of the enoylreductase activity, in particular, enoylreductase activity in the FAS protein complex comprising step of
  • the present invention provides an inhibitor of a ketoreductase, enoylreductase and/or MPT.
  • Said inhibitor may be an inhibitor containing all three inhibitory moieties inhibiting the enzymatic activities mentioned, namely, inhibiting the KR, ER and MPT activity.
  • the design of said inhibitor is such that the respective moieties can interact with the enzymatic counterparts. This is particularly true for an inhibitor of at least two of the enzymatic activities present in the FAS protein complex, e.g. the MPT an ER or MPT and KR as well as a combination of ER and KR or all three enzymatic activities.
  • the FAS protein complex may be a FAS protein complex with a gamma subunit of a recombinant polypeptide containing an insert while at the same time an inhibitor of at least one of the mentioned enzymatic activities is present.
  • further polypeptides of the present invention include polypeptides wherein at least one of the sequences or fragments thereof of Seq. ID No. 2, 3 and 4 are absent in a polypeptide of Seq ID No. 1.
  • Said polypeptides may contain additional segment(s) to maintain the structure of the underlying gamma-subunit.
  • the MPT inhibiting site is substituted with neutral, base and/or polar amino acid residues.
  • the present invention provides the inhibitor according to the present invention for use in the prophylaxis or treatment of cancer, infectious diseases, tuberculosis or besity, antibiotics against bacterial infections, in particular tuberculosis, or yeast infections.
  • the inhibitors of the present invention represents suitable inhibitors for treatment of diseases wherein the FAS protein complex is a target molecule for prophylaxis or treatment of said disease or disorder.
  • the inventors To mechanistically interrogate the structural determinants governing the iterative shuttling by yeast FAS, the inventors initially set out to establish a biochemical purification procedure that would yield highly active, biochemically and structurally homogenous FAS from the yeast S . cerevisiae. For this purpose, the inventors adapted the chromatography-free purification procedure the inventors had previously reported for 20S- and 26S proteasomes ( J. Schrader et al., Science. 353, 594-8 (2016 )) to the FAS. This approach relies on fractionated PEG precipitation and density gradient centrifugation as orthogonal purification steps e.g. se WO2017/211775 A1 .
  • Tma17p has previously been described to be involved in cellular translation or proteasome assembly ( T. C. Fleischer, et al., Genes Dev. 20, 1294-1307 (2006 )).
  • the co-purification and co-sedimentation of Tma17p with the FAS in the 40S region of the gradient suggests a direct biochemical interaction.
  • the inventors thus performed a series of control experiments to establish that Tma17p is indeed a bona fide interactor of the FAS. First, the inventors cross-validated the identification of the 20 kDa protein co-purifying with FAS as Tma17p.
  • the inventors generated a ⁇ tma17 knockout yeast strain and purified FAS from this strain using the same protocol as described above, obtaining comparable amounts of FAS.
  • the 20 kDa band is absent from the purified FAS fractions, corroborating the tandem-MS results.
  • the inventors investigated the biochemical stability of the interaction of Tma17p with FAS. For this purpose, FAS purified from a wild-type strain was loaded onto density gradients containing either 50 mM KCI, 150 mM KCI, or 250 mM KCI.
  • Tma17p quantitatively dissociates from the FAS at 250 mM KCI, which results in the sedimentation of Tma17p in the top fractions of sucrose density gradients, in the same way as recombinant Tma17p.
  • Tma17p quantitatively dissociates from the FAS at 250 mM KCI, which results in the sedimentation of Tma17p in the top fractions of sucrose density gradients, in the same way as recombinant Tma17p.
  • 150 mM KCI roughly half of the Tma17p co-purified with FAS was dissociated, whereas at 50 mM KCI co-purified Tma17p remained firmly associated to FAS and co-sedimented along with FAS in the 40S region.
  • the inventors investigated whether addition of recombinant Tma17p to ⁇ Tma17p-FAS could reconstitute the FAS-Tma17p complex.
  • Tma17p-FAS was incubated with increasing amounts of recombinant, fluorescently labelled Tma17p and analyzed by native gel electrophoresis on agarose gels, followed by quantification of fluorescence of FAS-bound Tma17p.
  • labelled Tma17p migrated close to the front, whereas FAS migration was considerably retarded owing to its native molecular weight of 2.6 MDa. Binding of Tma17p to FAS results in the shift of its mobility to a position where it co-migrates with FAS.
  • Tma17p is a weakly associated, integral subunit of the yeast FAS.
  • Tma17p is widely conserved in yeasts and appears to have escaped identification as a FAS subunit due to the relatively high-salt conditions previously employed in the art to purify FAS.
  • the inventors will refer to Tma17p as the FAS ⁇ -subunit and FAS bound to the ⁇ -subunit as the FAS holoenzyme.
  • the gamma subunit stabilizes a rotated FAS conformation
  • Tma17p constitutes an authentic ⁇ -subunit of FAS
  • the structure was solved by molecular replacement using the 3.1 ⁇ S . cerevisiae structure previously reported ( M.
  • the inventors were also able to obtain a ⁇ Tma17p-FAS structure by cryo-EM from 144,526 particles at global resolutions, estimated using the 0.143 FSC criterion, of 2.9 ⁇ with applied D3 symmetry, 3.0 ⁇ with applied C3 symmetry and 3.3 ⁇ in asymmetric reconstructions, with small local resolution differences.
  • the ⁇ Tma17p-FAS crystallographic model was refined against all three EM maps and resulted in models with an overall r.m.s.d. of 1 ⁇ with respect to the crystal structure, but less than 0.2 ⁇ r.m.s.d. amongst the different symmetric reconstructions.
  • the inventors then proceeded to determine the structure of the FAS holoenzyme by reconstituting ⁇ Tma17p-FAS with the recombinant ⁇ -subunit as described above in Figure 3 .
  • the inventors were able to reconstruct a cryo-EM structure from 110,597 single particles at a global resolution, estimated using the 0.143 FSC criterion, of 2.8 ⁇ with applied D3 symmetry, 2.9 ⁇ with applied C3 symmetry and 3.2 ⁇ in asymmetric reconstructions with small local resolution differences.
  • the quality of the FAS holoenzyme cryo-EM structure was comparable to that of the ⁇ Tma17p-FAS structure and allowed equally reliable model building.
  • the inventors solved the crystal structure of the FAS holoenzyme by molecular replacement using the ⁇ Tma17p-FAS crystal structure described above, which allowed for the reliable modelling of a C ⁇ -trace.
  • Models of the FAS holoenzyme refined against all three (symmetric and asymmetric) cryo-EM maps resulted in models with an overall r.m.s.d. of 0.8 ⁇ with respect to the crystal structure, but less than 0.2 ⁇ r.m.s.d. amongst the different symmetric reconstructions.
  • the similarity amongst the models derived from cryo-EM and X-ray crystallography of both ⁇ Tma17p-FAS and the FAS holoenzyme enables a reliable and conclusive interpretation of the structural impact of ⁇ -subunit binding.
  • XL-MS revealed an interaction of the N-terminal part of the ⁇ -subunit with the ER domain, a spatial proximity of the central part of the ⁇ -subunit to the MPT domain, and an association of the C-terminal part of the ⁇ -subunit with the KR and KS domains.
  • the XL-MS results and structural features corresponding to side-chain densities in the cryo-EM map, the inventors were able to build an atomic model for the ⁇ -subunit associated with FAS.
  • the inventors analyzed the steady-state kinetics and measured the rates of substrate turnover of ⁇ Tma17p-FAS with respect to varying concentrations of all substrates, specifically acetyl-CoA, malonyl-CoA and NADPH.
  • This analysis revealed saturation kinetics for both acetyl-CoA and malonyl-CoA, the binding of which were partially inhibited by the addition of saturating amounts of the ⁇ -subunit. Binding of the ⁇ -subunit resulted in a 2-fold decreased affinity for acetyl-CoA and a 4-fold decreased affinity in the case of malonyl-CoA.
  • the ⁇ -subunit partially impacts the enzymatic activity within the FAS holoenzyme, in particular the reductase activities, such that the dynamic range of NADPH substrate dependence is broadened to accommodate differences in cellular metabolism. This would allow cells to regulate FAS activity over an enhanced concentration regime of NADPH and NADP + .
  • the structural data described herein indicate that binding of the ⁇ -subunit impacts the conformational landscape of the FAS megaenzyme.
  • a rotated, compacted conformation with axially positioned ACP domains is favored in the presence of the ⁇ -subunit, while a non-rotated conformation with equatorially located ACP domains is adopted in its absence.
  • the inventors first examined by EM whether titration of the ⁇ -subunit into ⁇ Tma17p-FAS would cause an increase in the fraction of single particles adopting the rotated, compacted conformation.
  • the inventors collected several cryo-EM datasets of the FAS holoenzyme reconstituted with different amounts of the ⁇ -subunit. The inventors then refined 3D structures from each individual dataset, and analyzed the relative abundance of particles corresponding to populations of either the non-rotated- or the rotated conformation. Quantification indicated a clear correlation of the rotated conformation with the amount of ⁇ -subunit used for reconstitution. While in the ⁇ Tma17p-FAS sample 2% of selected particles correspond to the rotated state, this increases to 88 % at a 6-fold excess and 95 % at a 12-fold molar excess of the ⁇ -subunit.
  • the inventors To assess the conformational changes induced by binding of the ⁇ -subunit in a quantitative manner, the inventors next applied their formerly established cryo-EM method of 3D classification combined with the calculation of energy landscapes ( D. Haselbach et al., Cell. 172, 454-458.e11 (2018 )). For this purpose, the inventors initially refined 3D structures of the individual datasets with a mask corresponding to the central wheel, which is indistinguishable in conformation in both the non-rotated and rotated structures. To gain insight into the conformational space occupied by the dome in each individual dataset, the inventors then performed a 3D-classification without alignment in batches of 6,500 particles each, relying exclusively on the alignment parameters of the central wheel previously obtained.
  • the individual 3D classes thus obtained were individually refined to ensure their validity and subsequently subjected to 3D principal component analysis (PCA).
  • PCA 3D principal component analysis
  • the first eigenvector of this 3D PCA describes the compaction of the dome, whereas the second eigenvector signifies the notable rotation of the dome.
  • the inventors Since the inventors have an exact knowledge of the number of particles in each 3D class, the inventors are able to map an energy landscape by performing a Boltzmann interpolation within a coordinate system that is defined by the first and second eigenvectors of the PCA.
  • the energy landscapes for each individual dataset recapitulate the observation that binding of the ⁇ -subunit and ensuing formation of the FAS holoenzyme results in the adoption of the rotated conformation.
  • the energy landscapes indicate that binding of the ⁇ -subunit results in the formation of an energy barrier restricting access to the non-rotated conformation.
  • yeast strains with genetic deletions of the ⁇ -subunit gene will be beneficial for the biotechnological production of fatty acids and biofuels by yeasts.
  • ⁇ -subunit was labelled with NHS-Rhodamine (Thermo Fisher Scientific) according to the manufacturer's recommendations, followed by passage through a desalting column to remove excess unreacted dye.
  • Different concentrations of NHS-Rhodamine labelled ⁇ -subunit were titrated against 15.38 pmol ⁇ Tma17p-FAS in assay buffer in a total reaction volume of 40 ⁇ l. The mix was incubated at 30 °C for 30 minutes.
  • FAS holoenzyme (0.52 mg) was loaded on linear 10-45% (w/v) sucrose gradients in purification buffer containing 10mM DTT supplemented with either 0, 100, or 200 mM KCI. The gradients were centrifuged at 120,000 x g for 16h at 4 °C and were harvested in 200 ⁇ l fractions. All fractions of the gradients at the three salt concentrations were then analyzed by SDS-PAGE.
  • EM grids were prepared using both ⁇ Tma17p-FAS and FAS holoenzyme complexes at a protein concentration of 0.5 mg/ml.
  • the particles were adsorbed to a continuous carbon film attached to a Quantifoil (3,5/1) (Quantifoil, Jena, Germany) grid for 2 min at 4 °C.
  • Quantifoil 3,5/1) (Quantifoil, Jena, Germany) grid for 2 min at 4 °C.
  • the grid was then transferred to a Vitrobot Mark IV (Thermofisher, Germany) plunge-freezer, where it was vitrified after blotting for 8 s at 4 °C and 100% humidity.
  • ⁇ -subunit titration experiments 1.5 mg/ml ⁇ Tma17p-FAS solution was used and recombinant, purified ⁇ -subunit was titrated in 0-, 2-, 6-, 24-, and 96-fold excess. The protein mix was incubated at 30 °C for 30 min and then kept at 4 °C. 4 ⁇ l of each sample was applied to freshly glow-discharged Quantifoil R1.2/1.3 holey carbon grids (Quantifoil Micro Tools, Jena, Germany) followed by plunge freezing as described above.
  • Particle selection was done using Gautomatch (https://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/). All subsequent image processing steps were performed with Relion 3.0 ( J. Zivanov et al., Elife. 7 (2018), doi:10.7554/eLife.42166 ), unless otherwise specified.
  • the extracted particles were subjected to 3 rounds of reference free 2D classification to remove bad/empty images.
  • the remaining particles were refined with a mask around the central wheel using D3 symmetry.
  • EMDB-1623 was low pass filtered to 30 ⁇ and used as reference for all 3D refinement and 3D classification procedures. The refined particles were then classified without alignment, with a mask around the two domes.
  • the conformation of the dome was consistent among the different classes obtained, however, it was well resolved only in one class which consisted of ⁇ 50-60% of the particles used for classification.
  • the particles from the 3D class displaying the best resolution were selected and used for one more round of 3D refinement and classification as performed before.
  • the data further classified into 3 main classes.
  • the particles belonging to their respective classes were refined resulting in structures with resolutions of 3.5 ⁇ or below. Even though all the 3 classes were at comparable resolutions, their local resolutions, especially in the dome region of the molecule were very different. Only one class displayed high resolution details throughout the whole molecule.
  • the particles belonging to this class were selected and used to perform CTF refinement (and particle polishing for the FAS holoenzyme dataset) followed by 3D refinement.
  • the final refinement step was performed by applying different symmetries (C1-, C3- or D3 symmetry) to check for any structural artefacts that might be introduced due to the applied symmetry.
  • the 3D volumes were normalized and filtered to 20 ⁇ .
  • the inventors then performed 3D Principle Component Analysis (PCA) to obtain the major modes of motion among the different samples.
  • PCA 3D Principle Component Analysis
  • the first ( e 1 ) and second ( e 2 ) eigenvectors which depict motions of compaction and rotation of the FAS dome, respectively, were selected for calculating conformational landscapes.
  • the linear factors of e 1 and e 2 calculated as per equation (1) contributing to the input 3D volumes represent the x and y coordinates of the landscapes whereas the z axis represents the free energies of each class.
  • ⁇ Tma17p-FAS in purification buffer containing 10% sucrose (w/v), 10 mM DTT and 0.01% (w/v) LMNG was crystallized at 7 mg/ml by mixing 1 ⁇ l protein + 1 ⁇ l crystallization buffer A in Crystalgen hanging drop diffusion plates (Jena Bioscience, Germany) over a 750 ⁇ l reservoir of crystallization buffer A.
  • a 7-fold molar excess of ⁇ -subunit in the buffer used for ⁇ Tma17p-FAS was added to a 7 mg/ml protein solution of ⁇ Tma17p-FAS and incubated for 30 minutes at 30 °C.
  • the reconstituted complex was then crystallized by mixing 1 ⁇ l protein + 1 ⁇ l crystallization buffer B in Chryschem sitting drop vapor diffusion plates (Hampton Research, Aliso Viejo, USA) over a 500 ⁇ l reservoir of crystallization buffer B.
  • the crystals took 3-7 days to grow at 18 °C and were usually around 150 x 200 x 200 ⁇ m in size.
  • the crystals obtained were transferred to 4 °C by gradually decreasing the temperature in a linear manner over 48 h using a Rumed® E100 incubator (Rubarth Apparate GmbH, Germany). Stabilization and dehydration procedures were performed as following: 1) The drop was unsealed and 2 ⁇ l of the reservoir solution was added to the drop.
  • crystal stabilization buffer (A for ⁇ Tma17p-FAS, B for FAS holoenzyme) was added. 2) 2 ⁇ l of crystal stabilization buffer containing 5% (v/v) ethylene glycol was added followed by removal of 2 ⁇ l of solution from the drop. Then 2 ⁇ l of crystal stabilization buffer containing 5% (v/v) ethylene glycol was again added to the drop. This procedure was repeated with crystal stabilization buffer containing 10% and then 20% (v/v) ethylene glycol. 3) The reservoir solution was exchanged for crystal stabilization buffer containing 25% (v/v) ethylene glycol and the drop was resealed. The crystals were then allowed to equilibrate by vapour diffusion in excess of 16 h.
  • the crystals were harvested in Spine Litholoops (Molecular Dimensions, Suffolk, UK or Jena Bioscience, Jena, Germany) mounted on magnetic pins, and vitrified by plunge-cooling in liquid nitrogen.
  • the data were collected on EMBL beamline P14 at the PETRA III storage ring (DESY, Hamburg, Germany) using a MD3 vertical-spindle diffractometer (EMBL and Arinax, Moirans, France) and an EIGER 16M detector (Dectris, Baden, Switzerland).
  • a white beam refractive lens transfocator and slits were used to obtain homogenous X-ray beams which matched the crystal dimensions ( J. Schrader et al., Science. 353, 594-8 (2016 )).
  • Diffraction data were scaled and integrated with the XDS program package ( W. Kabsch, IUCr, XDS. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 125-132 (2010 )).
  • the refined ⁇ Tma17p-FAS crystal structure was used to determine the initial phases by molecular replacement with MOLREP for the FAS-holoenzyme diffraction data.
  • the obtained density map was found to correlate better with the FAS holoenzyme model (as determined by cryo-EM) rather than the ⁇ Tma17p-FAS structure. Therefore, the inventors used the model of ⁇ - and ⁇ -subunits from the FAS holoenzyme cryo-EM model in initial refinement of the X-ray data. Notably, the ACP domain and the ⁇ -subunit were removed from the structure prior to refinement to avoid introducing a phase bias.
  • the crystallographic ⁇ Tma17p-FAS model was used as the initial model for both the ⁇ Tma17p-FAS and FAS holoenzyme structures determined by cryo-EM. It was docked into the EM densities as a rigid body using UCSF Chimera, see above. An additional round of rigid body refinement was then performed in Refmac5. The model then underwent several rounds of manual modelling in Coot and refinement in Refmac5. Several models for the ⁇ -subunit were generated based on sequence homology using the Robetta ( D. E. Kim, et.al., Nucleic Acids Res. 32, W526-31 (2004 )) prediction server.
  • the inventors used the maps reconstructed using D3 symmetry as they had the best resolution.
  • Fourier shell correlation (FSC) between the refined model and the map was calculated (FSC sum ).
  • the model was also refined using the first unfiltered half-map. This model was then compared to the first half map (FSC work ) as well as to the second half-map (FSC free ) to check for signs of overfitting. All figures were made using Pymol (L. Schrödinger, Version 1.8" (2015)), Chimera and ChimeraX ( T. D. Goddard et al., Protein Sci. 27, 14-25 (2018 )).
  • Standard chemicals were obtained from Sigma Aldrich (Taufmün, Germany). Detergents were obtained from Anatrace (Maumee, USA), crystallization plates from Hampton Research (Aliso Viejo, USA) and Jena Bioscience (Jena, Germany), Litholoops from Molecular Dimensions (Suffolk, UK) and Jena Bioscience (Jena, Germany). A Rumed® E100 incubator from Rubarth Apparate GmbH (Hannover, Germany) was used to transfer crystallization plates to 4 °C. EM grids were from Quantifoil (Jena, Germany). S.
  • cerevisiae cells were grown in a 250 L fermentor from Infors GmbH (Delitzsch, Germany), yeast cells were ground in a ZM200 mill from Retsch (Haan, Germany), E. coli cells were disrupted using an Emulsiflex C3 fluidizer from Avestin (Mannheim, Germany) and fluorescence from native gels was measured using an Amersham imager 600 (GE Healthcare, Kunststoff, Germany).
  • Saccharomyces cerevisiae strains BJ2168 (MATa prc1-407 prb1-1122 pep4-3 leu2 trp1 ura3-52 gal2) and tma17 ⁇ BJ2168 (MATa prc1-407 prb1-1122 pep4-3 leu2 trp1 ura3-52 gal2 tma17::kanMX) were used in this study.
  • Cells were grown in YPD medium in an Infors 250 litre fermenter and harvested in late log phase at an OD 600 of 9-10.
  • cells were washed with cold ddH 2 O and then resuspended in 2X purification buffer containing 20% (w/v) sucrose such that 2 ml of buffer was added per gram of cells. Cells were then flash frozen as beads in liquid nitrogen and stored at -80 °C until further use.
  • the purification strategy was adapted from protocols developed earlier for the purification of human 20S and 26S proteasomes ( D. Haselbach et al., Nat. Commun. 8, 1-8 (2017 )).
  • the ground powder was thawed in a water bath at 37 °C, supplemented with purification buffer to 0.33x concentration from a 10x stock, followed by the addition of sucrose powder to 20% (w/v), benzamidine chloride to 10 mM and PMSF to 1 mM (from a 100 mM stock solution in propanol).
  • the extract was incubated at 25 °C on a magnetic stirrer for 30 minutes followed by centrifugation at 30,000 x g for 30 minutes at 4 °C. After centrifugation, the supernatant was filtered through 3 layers each of cheese cloth and miracloth to obtain a S30 yeast cell extract.
  • Octyl Glucose Neopentyl Glycol (OGNG) (from a 10% (w/v) stock) was added to a final concentration of 0.2% (v/v) and the extract was incubated at 30 °C for 30 minutes followed by centrifugation at 100,000 x g for 1 hour at 4 °C. The supernatant was again filtered through 3 layers each of cheese cloth and miracloth.
  • PEG400 P oly E thylene G lycol400
  • number signifies the mean molecular weight of the PEG polymer
  • PEG400 was added to a concentration of 20% (v/v) to the yeast S100 extract while stirring at 18 °C and incubated for 30 minutes.
  • Precipitated proteins were removed by centrifugation at 30,000 x g for 30 minutes at 4 °C.
  • the supernatant was then precipitated by raising the concentration of PEG400 to 30% (v/v) as described above.
  • the precipitate of this step which contains the FAS, was recovered by centrifugation at 30,000 x g for 30 minutes at 4 °C and resuspended in purification buffer containing 2% (w/v) sucrose, 10 mM DTT and 0.01% (w/v) Lauryl Maltose Neopentyl Glycol (LMNG) in an orbital shaker at 18 °C.
  • the resuspended material was loaded on 10-45% (w/v) linear sucrose gradients in purification buffer containing 10 mM DTT, which were centrifuged at 100,000 x g for 16 h at 4 °C. Gradients were harvested in 1 ml fractions. SDS-PAGE was utilized to identify fractions containing FAS.
  • the ⁇ -subunit was cloned as a synthetic gene into a pET151/D-TOPO® plasmid (Geneart, Regensburg) and expressed with an N-terminal His 6 -TEV-tag in BL21 Star (DE3) competent cells.
  • the transformed cells were grown at 37 °C with 180 rpm shaking until an OD 600 of 0.5 was reached, the temperature was then decreased to 18 °C and expression of the ⁇ -subunit was induced 1 h after reduction of the temperature by the addition of 0.5 mM IPTG.
  • ⁇ -subunit was expressed for 16 h at 18 °C and 180 rpm. The cells were harvested by centrifugation (5000 x g, 15 minutes, 4 °C), washed with cold ddH 2 O and stored at -80 °C.
  • the obtained lysate was centrifuged (30 min, 50,000 g, 4 °C) and filtered through Minisart NML Plus cellulose-acetate syringe filters with a 0.45 ⁇ m pore size (Sartorius).
  • the cleared lysate was loaded onto a Ni-NTA gravity column (5 ml bed volume) pre-equilibrated with 20 column volumes (CV) of resuspension buffer.
  • the column was washed with 20 CV resuspension buffer, followed by 20 CV of washing buffer (20 mM Tris-HCI pH 8.0, 500 mM NaCl, 20 mM imidazole, 0.2 mM PMSF, 10 mM benzamidine).
  • elution buffer (20 mM Tris-HCI pH 8.0, 500 mM NaCl, 500 mM imidazole, 0.2 mM PMSF, 10 mM benzamidine) were applied and collected in 2 ml fractions.
  • TEV-protease was added to the protein in an enzyme-to-protein ratio of 1:50 followed by dialysis overnight in 5 I dialysis buffer (20 mM Tris-HCI pH 8.0, 5 mM ß-ME, 100 mM NaCl) at 4°C.
  • the digested ⁇ -subunit was applied to a Ni-NTA column, which was pre-equlilibrated with 20 CV dialysis buffer. The flow-through was collected in 2 ml fractions and single fractions were analyzed by SDS-PAGE.
  • Fractions containing pure ⁇ -subunit were pooled and concentrated to a final concentration of 20 mg/ml using a 10 kDa MWCO Amicon Ultra 15 centrifugal concentrator (Millipore). The final protein was aliquoted, frozen in liquid nitrogen and stored at -80 °C.
  • UV-absorption was detected at 215 nm, 260 nm and 280 nm, and purification was performed with a linear gradient of A (water + 0.1% (v/v) TFA) to B (79.9% acetonitrile (v/v) + 20% ddH2O + 0.1% (v/v) TFA).
  • the peak fractions of the corresponding substrates were collected, lyophilized and stored at -80 °C until use.
  • the enzymatic reaction was started by addition of malonyl-CoA and measured at 30 °C.
  • the obtained data were fitted with different kinetic models including the Michaelis-Menten equation, the Hill equation or with a modified Michaelis-Menten equation implementing substrate activation.
  • Peptides were acidified with trifluoroacetic acid (TFA) added to a final concentration of 0.5% (v/v), desalted on MicroSpin Colums (Harvard Apparatus) following the manufacturer's instructions, and vacuum dried. Peptides were resuspended in 50 ⁇ L of 50% acetonitrile/0.1% (v/v) TFA before enriching of cross-linked species by peptide size exclusion (SuperdexPeptide 3.2/300 column, GE Healthcare). Fractions of 50 ⁇ L were collected and those that eluted first and contain the cross-linked peptide pairs were analyzed by LC-MS/MS.
  • TFA trifluoroacetic acid
  • the gradient started at 5% B, increasing to 12, 15 or 20% B within 3 min (according to fraction), followed by a continuous increase to 48% B within 45 min, then keeping B constant at 90% for 8 min. After each run the column was again equilibrated to 5% B for 2 min.
  • the flow rate was set to 300 nL/min.
  • MS1 survey scans were acquired in the orbitrap (OT) with a resolution of 120,000, an injection time (IT) of 60 ms and an automatic gain control (AGC) target of 5 ⁇ 10 5 .
  • Dynamic exclusion (DE) was set to 10 s and only charge states between 3 and 8 were considered for fragmentation.
  • MS2 spectra were acquired in the OT of the 20 most abundant precursor ions, resolution 30,000, IT 120 ms and AGC target 5 ⁇ 10 4 . Fragmentation was enforced by higher-energy collisional dissociation (HCD) at 30% normalized collision energy (NCE). The second replicate was treated with the following changes: we used a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific), at MS1 the IT was set to 50 ms and the AGC target to 1 ⁇ 10 6 . At MS2, the IT was set to 128 ms and the AGC target to 1 ⁇ 10 5 . DE covered 30 ms. Here, the 30 most abundant precursor ions were considered for fragmentation.
  • HCD collisional dissociation
  • NCE normalized collision energy
  • ProteomeDiscoverer 1.4 (Thermo Fisher Scientific) was used for converting .raw files into .mgf format (signal-to-noise ratio 1.5, 1000-10000 Da precursor mass).
  • the generated .mgf files were subjected to pLink v. 1.23 ( B. Yang et al., Nat. Methods. 9, 904-906 (2012 )) to identify cross-linked peptides.
  • pLink v. 1.23 B. Yang et al., Nat. Methods. 9, 904-906 (2012 )
  • FDR was set to 0.01.
  • Spectra of inter-cross-links between FAS and TMA17 within the first replicate were evaluated manually.
  • a score cut-off of 3 ( - log 10 transformation of the original pLink score) was applied and only cross-links supported by more than one cross-linked peptide spectrum match (CSMs) were considered.
  • CSMs cross-linked peptide spectrum match

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